Efficient and multiplex gene upregulation in plants through CRISPR/Cas-mediated knock-in of enhancers

Gene upregulation through genome editing is important for plant research and breeding. Targeted insertion of short transcriptional enhancers (STEs) into gene promoters may offer a universal solution akin to transgene-mediated overexpression, while avoiding the drawbacks associated with transgenesis. Here, we introduce an "in-locus activation" technique in rice that leverages specifically screened STEs for refined, heritable, and multiplexed gene upregulation. To address the scarcity of potent enhancers, we developed a large-scale mining approach and discovered a suite of STEs capable of enhancing gene expression in rice protoplasts. The in-locus integration of these STEs into eight rice genes resulted in substantial transcriptional enhancements, with up to 869.1-fold increases in the edited plants. Employing a variety of STEs, we achieved delicate control of gene expression, enabling the fine-tuning of key phenotypic traits such as plant height. Our approach also enabled efficient multiplexed gene upregulation, with up to four genes simultaneously activated, significantly enhancing the nicotinamide mononucleotide (NMN) metabolic pathway. Importantly, heritability studies from the T0 to T3 generations confirmed the stable and heritable nature of STE-driven gene activation. Coupled with our STE-mining technique, in-locus activation holds great promise to make gene upregulation a major application of genome editing in plant research and breeding.